Congenital heart defects (CHDs) are the leading cause of death in infants and young children and those suffering from congenital diaphragmatic hernia and associated CHDs (particularly with left heart hypoplasia (LHH)), have high mortality. The dominant hypothesis is that the CHDs result from mechanical factors leading to altered blood flow in the left heart, such as compression of the left heart by visceral structures protruding into the thoracic cavity. In mild LHH, left ventricular dimensions tend to normalize after birth and hernia repair, further implicating the role of altered mechanical loads in certain forms of CHD. Decreased cardiac tropoelastin and procollagen gene expression and decreased left ventricular function in LHH compared to normal hearts suggest that significant deviations from normal ECM composition and mechanical properties of the myocardium occur. Given that ECM properties can affect various cell functions including proliferation, differentiation, and phenotype, any deviations from the normal composition and stiffness of the ECM in the progression of CHD would have a significant impact on the myocytes, resulting in profound alterations to the development and function of the myocardium. Additionally, altered flow through the developing heart will affect mechanical strain in the ventricular wall, and studies have demonstrated that changes in mechanical strain can significantly impact cell function. We hypothesize that by mimicking changes in ECM composition, stiffness, and/or mechanical strain associated with LHH, we will be able to guide embryonic myocytes towards the LHH phenotype while mimicking the healthy embryonic heart environment will lead to normal myocyte development. In Aim 1 we will use nitrofen-induced congenital diaphragmatic hernia in developing rat embryos to generate models of CHD. Healthy and diseased hearts from embryonic and fetal stages will undergo decellularization to obtain cardiac ECM which will be assayed to determine any compositional differences and alterations in mechanical stiffness. At these same life stages, we will characterize native myocyte proliferation and maturation in healthy and CHD hearts. We will then culture embryonic/fetal myocytes isolated from healthy and CHD ventricles in rationally altered 2D environments that mimic different combinations of healthy and diseased biophysical properties using an ECM-coated polyacrylamide (PA) gel system and assess whether stiffness and composition act synergistically or antagonistically, and whether healthy biophysical cues can drive diseased myocytes towards a healthy phenotype. In Aim 2, we will subject embryonic and fetal myocytes to mechanical strain of varying amplitude and frequency and assess the proliferation, maturation, and function of myocytes from healthy and CHD hearts. These studies are novel and will represent one of the first experiments to assess the role of altered biophysical and biomechanical signaling in the development of cardiac pathologies during growth in utero. PUBLIC HEALTH RELEVANCE: This research involves the study of changes to the structure and function of the hearts connective tissue following the development of a congenital heart defect. Understanding how these changes affect the muscle cells of the heart could lead to new and improved therapies for treating congenital heart defects, which are the leading cause of death for infants and young children. The proposed research aims to develop an understanding of how heart cells sense the environment during abnormal heart development and seeks to exploit this information to develop new methods for pediatric cardiac repair.